mycotoxins contamination in animal food and feed ... · pdf filemycotoxin contamination of...

International Journal of Science and Research (IJSR) ISSN (Online): 2319-7064

Index Copernicus Value (2013): 6.14 | Impact Factor (2013): 4.438

Volume 4 Issue 5, May 2015

www.ijsr.net Licensed Under Creative Commons Attribution CC BY

Mycotoxins Contamination in Animal Food and

Feed

Ashok K. Shukla

Assistant Prof. Dept. of Microbiology & Biotechnology, Holy Cross Women’s College, Ambikapur, Chhattisgarh, India

Abstract: Many fungi, both field and storage fungi produce metabolites that are poisonous, sometimes fatal, to men and animals.

Production of these mycotoxins depends on species or strain of the fungus and on the ecological conditions for its development, in

particular food source, moisture, temperature, mechanical damage, hot spots, time duration, O2 concentration, CO2 level, nature of

substrate mineral nutrition, chemical treatment, plant stress, invertebrate vectors, fungal inoculums, fungal strain differences and

interaction of different microorganisms. Grains those are spoiled and unfit for human consumption or having poor quality are generally

used for making animal food and feed. There are now over 400 recognized mycotoxins found in feed material and it has been reported

that as much 25% of the world’s cereal grains may be contaminated with mycotoxins. Mycotoxins are toxic, chemically diverse

secondary substances or metabolites produced by wide range of fungi including Aspergillus, Penicillium, Fusarium, Trichothecium,

Claviceps sps. etc. recognition of the symptoms of mycotoxins poisoning in an affected animal can be quite difficult to do confidently

because many of the symptoms of chronic but sub acute cases are the same as those resulting from a wide range of causes. Common

symptoms include: rough coat, poor body condition, low milk yield with poor quality milk components, foot lesions, poor reproductive

performance, an increase incidence of abortion and inconsistent much with mucus tags. In present investigation 8 different compositions

such as khali containing alsi; khali (mustard); chuni(arhar dal); chuni (urad dal), rice, wheat and gram bran mixture; hay and fine rice

chaffs were collected from various storage conditions and animal feed centre and were tested for mycotoxin contamination. Isolation of

associated fungi was made in Czapak Dox Agar, Malt Extract Agar and Potato Dextrose Agar media. More than 53 isolates belonging to

15 different genera were islolated and are : Aspergillus flavus, A. candidus, A. terreus, A. glacus, A. fischeri, A. nidulans, A. niger,

Absisia coryambifera, Alteranaria alternate, Cladosporium sp., Fusarium sp., Humicola sp., Mucor pusillus, Rhizopus sp. and

Trichohecium roseum. Mycotoxin contamination recorded on the basis of the fungi as listed by Jelinek et al (1989). At about 70%

samples were found with positive mycotoxin contamination.

Keywords: Mycotoxins, Animal food and feed, Aspergillus, Penicillium

1. Introduction

Animal feed is any agricultural foodstuff used specifically

to feed domesticated livestock such as cattle, goats, sheep,

horses, chickens and pigs. Cattle feed is a concentrate,

basically comprising cereals and usually by-products of

plants and animal sources and this concentrate is then

mixed with green fodder. Due to high feed cost, mixing of

stale bread, kitchen and bakery wastes in the feed are in

practice. These waste products are usually tainted with

fungus and may be a contributing factor in mycotoxin

production in cattle feed.

“Mycotoxins are those secondary metabolites of fungi

that have the capacity to impair animal health and

productivity (D'Mello and Macdonald, 1998)”. There

are over 100 species of fungi that can infect plants and

produce mycotoxins. Molds can infect dairy cattle,

especially during stressful periods when they are immune

suppressed, causing a disease referred to as a mycosis. The

diverse effects precipitated by these compounds are

conventionally considered under the generic term

"mycotoxicosis", and include distinct syndromes as well as

non-specific conditions. Mycotoxins can be formed on

crops in the field, during harvest, or during storage,

processing, or feeding. Molds are present throughout the

environment. Spore concentrations are high in the soil and

in plant debris, and lie ready to infect the growing plant in

the field.

Mycotoxin contamination of forages and cereals

frequently occurs in the field following infection of plants

with particular pathogenic fungi or with symbiotic

endophytes. Contamination may also occur during

processing and storage of harvested products and feed

whenever environmental conditions are appropriate for

spoilage fungi. Moisture content and ambient temperature

are key determinants of fungal colonization and mycotoxin

production. It is conventional to subdivide toxigenic fungi

into "field" (or plant-pathogenic) and "storage" (or

saprophytic/spoilage) organisms. Claviceps,

Neotyphodium, Fusarium and Alternaria are classical

representatives of field fungi while Aspergillus and

Penicillium exemplify storage organisms. Mycotoxigenic

species may be further distinguished on the basis of

geographical prevalence, reflecting specific environmental

requirements for growth and secondary metabolism. Thus,

Aspergillus flavus, A. parasiticus and A. ochraceus readily

proliferate under warm, humid conditions, while

Penicillium expansum and P. verrucosum are essentially

temperate fungi. Consequently, the Aspergillus

mycotoxins predominate in plant products emanating from

the tropics and other warm regions, while the Penicillium

mycotoxins occur widely in temperate foods, particularly

cereal grains. Fusarium fungi are more ubiquitous, but

even this genus contains toxigenic species that are almost

exclusively associated with cereals from warm countries.

Mycotoxins can be the primary agent causing acute health

or production problems in a dairy herd, but more likely,

mycotoxins are a factor contributing to chronic problems

Paper ID: SUB154163 712





including a higher incidence of disease, poor reproductive

performance or suboptimal milk production.

2. Materials and Method

2.1 Sampling: Sample was collected using given

methods (Shukla,2015)

Collection of fodder samples: Various fodder samples

khali(alsi –Linum asitatissimum) , khali(mustard –

Brassica juncea & B.nigra), rice bran (Oryza sativa), wheat

bran(choker Triticum aestivum), chuni containing pulses

powder such as arahar dal (Cajanus cajan), chuni

containing urad dal (Vigna mungo), chuni containing

masoor dal (Lens culinaris) and fine rice chaffs (Oryza

sativa)] were collected from various storage conditions and

animal feed centers with the help of sterilized spatula in

sterilized transparent polythene bags. The bags containing

sample were sealed and brought to the laboratory for

further use.

Sample storage: Collected samples were stored at 4oC in

refrigerator, to provide low temperature, low moisture

content, low relative humidity storage conditions until they

were analysed.

2.2 Analysis: Analysis was done under following:

Isolation of fungi associated with different cattle feeds:

small amount of sample was inoculated on prepoured

plates of Malt Extract Agar medium plates and Czapek’s

Dox Agar medium plates. Antibiotics like penicillin and

streptomycin were added in medium @30 µg/l to prevent

bacterial growth.

Incubation: Inoculated plates were kept in incubator at 28o

C and 45o C for 72 to 96 hrs.

Identification of microorganisms: for identification of

microorganisms fungal smear was prepared and was

observed under research microscope and identification was

done with the help of available literature and monograph.

(Shukla, 1991; Shukla, 2014 d,e)

2.3 Analysis of Mycotoxin: Mycotoxin

contamination in various foods is a major threat to health

of exposed people. Therefore, mycotoxin analysis is an

important subject of many investigations. For the study,

following procedure was followed.

Fungi isolated from feed samples namely Aspergillus

flavus, A. paraciticus, A. fischerii, A. candidus , A.

terreus, A. glacus, A. fischeri, A. nidulans, A. niger, T.

roseum and Fusarium culmorum directly inoculated on

conical flask containing 150 ml Malt Extract broth

medium.

Incubation: Inoculated flasks were kept for incubation in

BOD incubator at temperature of 28o C for 7 days.7 days

later Fungal mat with thick mycelial growth was appeared

in the flask.

2.4 Procedure for calculation of dried weight of

fungal mycelium

Whatman filter paper number 42 was kept in hot air oven

at 105o C for 24hrs.After 24hrs, the weight of filter paper

was taken using an electronic balance.bFungal mat was

filtered from the medium using sterilized pre weighed

Whatman filter paper and funnel and filterate was

preserved for further use. Filtered mycelium was

compressed for removal of extra liquid. Thereafter, filter

paper containing mycelium was dried in hot air oven and

the dried mycelium mass was weighed using an electronic

balance (Shukla, 2014e).

Dry weight of mycelium= (weight of filter paper

containing mycelium – weight of filter paper)

2.5 Extraction of mycotoxin

The filtrate obtained previously was measured using

measuring cylinder and in equal proportion to it ethyl

acetate was taken. Both the filtrate and ethyl acetate was

taken in a separator funnel and was shaken well upto 30

minutes. Kept for standing for 1 hr and two separate layers

was observed. Using the separator funnel upper layer was

separated and preserved which conatained ethyl acetate

and mycotoxin and lower layer was discarded.

Crude mycotoxin preparation: In waterbath mycotoxin

containing solution was boiled at the boiling point of ethyl

acetate. The solution was reduced up to 1-2 ml. The

obtained solution is the crude mycotoxin. To analyzed the

presence of mycotoxin using thin layer chromatography to

separate the toxins present in the crude mixture previously

obtained.

Firstly, to the TLC plates silica gel mixture (silica gel &

water) was spread like a thin film of approx 5mm

thickness and was air dried. Thus the solid phase was

prepared. (Xie,2009 ; Stahl, 1953 and Shukla 1991)

Crude mycotoxins sample was loaded on the TLC plate

using a capillary tube.

Solvent system for toxin determination contained

chloroform and methanol (95:5 v/w) this solvent was put

into the TLC chamber and then sample loaded TLC plates

were kept into the chamber, so that the mobile phase can

run over solid phase separating out the toxins.

After 2 hrs the plates were taken out of the solvent system

and were air dried.

The air dried plates were observed under the UV chamber

for the presence of toxins. If toxin was present in the

sample then the bands of toxin was observed under the uv

chamber.

3. Results and Discussion

Cattle feed are a complete nutrient diet for cattles having

all the source of nutrients such as protein, carbohydrate

and fats. On the analysis of animal feed sample in culture






medium a wide variety of fungi were detected. In our

investigation we have taken 8 samples namely [khali(alsi),

khali(mustard), rice bran, wheat bran(chokar), chuni

containing arahar dal, chuni containing urad dal, chuni

containing masoor dal and fine rice chaffs] were tested for

fungal contamination.

Table 1: Occurrence of fungi in various samples of animal food (Shukla, 2015; Shukla, 2014c)

Name of

Microorganisms

Khali

(alsi)

Khali

(musterd)

Chuni (arahar

dal)

Chuni

(urad

dal)

Rice

Bran

Wheat

Bran

Chuni

(masoor

dal)

Fine

rice

chaff

Aspergillus flavus + - + + - + + +

A. candidus - - - - + + + -

A. paraciticus - + + - - + + +

A. fiscerii + - - - - + - -

A. fumigates + + + - + - + +

A. glacus - - - - - + + -

A. nidulans - + + - + + + -

A. terreus - - - + + + + -

F. culmorum - - + - - + + +

R rhizopodiformis + + + + + + - -

R. oryzae + - - - + + + +

Mucor pusillus - + - - - + + -

Alternaria alternate + + - - + + + -

Cladosprium + + + + + - - +

T. roseum - - - - + + + -

H. terricola - - + + + + - +

We found that the common fungi which occurred in the

feed samples were Aspergillus flavus, Aspergillus

paraciticus, Aspergillus candidus, Aspergillus fischeri, A.

nidulans, A. glocus, A.terreus, Hmicola sp., Trichothecium

roseum Fusarium culmorum and Rhizopus species. Rice

bran and wheat bran had high fungal contamination

whereas Khali (alsi), Khali mustered & Fine Rice chaff

had only five fungi are appeared and showed lowest fungal

diversity (Table 1).

It is generally found that animal feeds are highly

contaminated with mycotoxins due to poor handling and

poor storage conditions. Identified species of fungi

(Aspergillus flavus, Aspergillus paraciticus, Aspergillus

candidus, Aspergillus fischeri, Aspergillus glocus,

Aspergillus ocheracius, Aspergillus nidulans, Aspergillus

terreus Trichothecium roseum, and Fusarium culmorum)

were further inoculated for mycotoxin production in Malt

extract broth. After 7 days of incubation the dry weight of

fungal mycelium was noted (Table 2).

Table 2: Mycotoxin production by fungi isolated from animal food

S. No Name of

microorgsnism

Wet weight of the mycelium

(gm)

A

Total water content

(gm)

B

Dry mycelium weight

(gm)

(A-B =C)

Mycotoxin

production*

1 A. flavus 1.32 0.960 0.360 +++

2 A. paraciticus 1.21 0.962 0.248 ++

3 A. candidus 1.20 0.964 0.236 +++

4 A. fischeri 1.28 0.961 0.319 -

5 A. glocus 1.23 0.963 0.267 ++

6 A. ocheracius 1.17 0.964 0.206 ++

7 A. nidulans 1.44 0.967 0.473 ++

8 A. terreous 1.28 0.966 0.314 ++

9 F. culmorum 1.14 0.963 0.177 -

10 T. roseum 1.26 0.965 0.295 +






*indicates the visual intensity of spots on TLC plates such

as +++strong,++moderate, +poor production of mycotoxin

and -indicates no mycotoxins production.

Detection of mycotoxin presence was done with the help

of TLC. Mycotoxin producers gave fluorescent bands

when observed under UV lamp of Wavelength 365nm.

Figure 1: A. flavus & A. paraciticus showing bands of toxin when viewed under UV light

On the basis of visual intensity of bands, the level of

mycotoxin was detected. It was found that A. candidus and

Aspergillus flavus produced the highest level of mycotoxin

and Trichothecium roseum produced the lowest whereas

Fusarium culmorum and Aspergillus fischeri shows no

production. Also it was found that about 70% of the feed

samples were found with positive mycotoxin

contamination. Hence, the amount of contamination level

is high which will cause the adverse effects to animals.

4. Conclusions

Animal feeds are routinely subject to contamination from

diverse sources, including environmental pollution and

activities of insects and microbes. Animal feeds may also

contain endogenous toxins arising principally from

specific primary and secondary substances produced by

fodder plants. Thus, feed toxins include compounds of

both plant and microbial origin. The FAO has estimated

that worldwide about 25% of crops are affected annually

with mycotoxins. Such surveys reveal sufficiently high

occurrences and concentrations of mycotoxins to suggest

that mycotoxins are a constant concern. Every year a

significant percentage of the world's grain and oilseed

crops is contaminated with hazardous mycotoxins such as

aflatoxins. Detection, removal and diversion are

reasonable means for preventing the entry of mycotoxins

into the food chains. The best way of controlling

mycotoxin contamination is by prevention and can be

accomplished by reducing fungal infection in growing

crops through the adoption of suitable cultural practices,

by rapid drying or by the use of suitable preservatives

(Sinha, 1993) If contamination cannot be prevented, a way

to either remove or destroy the toxin will allow

consumption of the commodities with reduced adverse

effect. Mycotoxins can be eliminated or detoxified by

physical, chemical or biological techniques. Many

chemicals including numerous acids, alkalis, aldehydes,

oxidizing agents and several gases have been tested for

their ability to degrade or inactivate aflatoxin and many

other mycotoxins. There is need to adopt effective

strategies for mycotoxin decontamination and mycotoxin

detoxification. The formulation and implementation of

mycotoxins regulatory limits, regular analysis of animal

feed and feed ingredients and employment of proper

mycotoxin decontamination and deactivation strategy will

help to reduce the economic losses to a great extent

(Shukla,2015).

References

[1] Allcroft, R. and R.B.A., Carnaghan. (1962).

Groundnut toxicity: Aspergillus flavus toxin

(aflatoxin) in animal products. Vet. Rec. 74:863-864

[2] CAST. Mycotoxins -Risks in plants, animal, and

human systems. 2003. Ames, Iowa, USA

[3] Dhand, N.K., Joshi, D.V. & Jand, S.K. (1998). Fungal

contaminants of dairy feed and their toxigenicity.

Indian Journal of Animal Sciences, 68: 1095-1096

[4] D'Mello, J.P.F. (2000). Antinutritional factors and

mycotoxins. In J.P.F. D'Mello, ed. Farm animal

metabolism and nutrition. Wallingford, UK, CAB

International. p. 383-403.

[5] D'Mello, J.P.F. (2001). Mycotoxins in plant products.

In J.P.F. D'Mello, ed. Food safety. Wallingford, UK,

CABI Publishing. (in press)

[6] Hamilton, P.B. (1984). Determining safe levels of

mycotoxins. J. Food Prot. 47:570-575

[7] Shukla, A. K.(1991). Spoilage of wheat and Sorghun

grain produced by thermophilus fungi during storage

Ph.D thesis, Dr. H S Gour Kondriya Vishvidyalaya

Sagar. PP 216-223.

[8] Shukla, A. K., Jain, P. (2009). Aflatoxin

contamination on wheat. Research zone. PP 11-13.

[9] Shukla, A.K. (1991). Spoilage of cereal grains by

thermophilous fungi. Vistas in seed biology, Vol.-1

pp352-365.

[10] Shukla, A.K. and Jain P.C.(1991). Thermophilous

mould flora: A major deteriogenic factor for wheat

grain. Proc. Nat. Sem. On advances in seed science

and tech. Pp 58-59

[11] Shukla, A.K. (2015) Deterioration of quality and

quantity of wheat grains during storage. IJSR, ISSN:

2319-7064, Volume-4, Issue-1, pp1758-1767. (Impact

factor- 4.438)

[12] Shukla, A.K. (2014a) Biodiversity in Aspergillus

nidulans group on the basis of lipases profile. IJSR,






ISSN: 2319-7064, Volume-3, Issue-6, pp1391-1394.

(Impact factor- 3.358)Paper ID-02014487

[13] Shukla, A.K. (2014b) Degradation of timber wood by

filamentous fungi. IJSR, ISSN: 2319-7064, Volume-

3, Issue-7, pp1091-1094. (Impact factor- 3.358)Paper

ID-020141184

[14] Shukla, A.K.(2014c) Extracellular production of

thermostable endoglucanase by thermophilous fungi.

IJSR, ISSN: 2319-7064, Volume-3, Issue-8, pp 110-

113. (Impact factor- 3.358)Paper ID-020151165

[15] Shukla, A.K. (2014d) Occurrence of keratinophilic

fungi from the soils of Chhattisgarh. IJSR, ISSN:

2319-7064, Volume-3, Issue-9, pp 2041-2044.

(Impact factor- 3.358)Paper ID- SEP14574

[16] Shukla, A.K. (2014e) Occurrence of mycotoxins in

storage fungi. IJSR, ISSN: 2319-7064, Volume-3,

Issue-11, pp 1747-1750. (Impact factor- 3.358)Paper

ID-OCT 14188

[17] Shukla, A.K. & Singh, Annu (2014) Diversity of

forest of Surguja District Chhattisgarh, India. IJSR,

ISSN: 2319-7064, Volume-3, Issue-6, pp1153-1157.

(Impact factor- 3.358)Paper ID-SUB 14653

[18] Stahl, E. (1965) Thin layer chromatography-a

laboratory hand book. Academi press, New York.

[19] Sinha, R.N. (1973) Ecology of storage. Annals de

tecnologie agricole. 22:351-361.

[20] Lacey, J. (1975) Moulding of grain in relation to

mycotoxins formation. International journal of

environmental studies. 8:183-186


mycotoxins contamination in animal food and feed ... · pdf filemycotoxin contamination of...

Documents